We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. Human GC-1 (hGC-1) is a novel olfactomedin-like glycoprotein that is expressed during granulocytic differentiation. To further characterize the function of GC-1, we have cloned the cDNA for the mouse homologue of human GC-1 and investigated the pattern of GC-1 gene expression in the mouse during embryonic development and in adult tissues. Genomic and cDNA clones corresponding to mouse GC-1 (mGC-1) were isolated. The mGC-1 protein shares 93% homology with hGC-1 at the amino acid level. The protein sequence of mGC-1 indicates that it belongs to the olfactomedin-related glycoprotein family, which includes olfactomedin, TIGR, NOELIN-2 and latrophilin-1. Fluorescence in situ hybridization (FISH) was used to map the mGC-1 gene locus to chromosome 14D3. Like other olfactomedin-like genes with tissue-restricted patterns of expression, mGC-1 is expressed at high levels in the small intestine and kidney, at moderate levels in the stomach, thymus, spleen, and no expression was detected in brain, heart, liver or lung. These results are consistent with the distribution of hGC-1 in human tissues. Analysis of mGC-1 expression by in situ hybridization in mouse embryos showed that mGC-1 is not expressed until day E15. mGC-1 expression was detected in the small intestine and stomach. Interestingly, a very clear demarcation of the mGC-1 signal was evident in the intestine, where mGC-1 was specifically expressed between the enterocytes lining the villi, but not in the lamina propria or in the muscularis layers. There was a similar, albeit weaker, pattern of mGC-1 expression in stomach. In situ hybridization showed mGC-1 is strongly expressed in the crypts of small intestine. hGC-1 cDNA has been transfected into 293 cells transiently and stably. In transfected 293 cells, hGC-1 was detected in the culture medium after 48h of transfection. In the Western blot, hGC-1 showed a multimer form in non-reducing gel, which was reduced to monomer after adding 10mM DTT. hGC-1 was also detected in the perinuclear region and cell surface.These data suggest hGC-1 is a secreted glycoprotein. This is consistent with tissue expression result that hGC-1 is only abundantly expressed in tissues which has secretion capability. On further analysis, we find high level expression of mGC-1 in pro-B and pre-B cells and low-level expression in mature B and T-cells that co-localizes in this region of the small intestine. When the myeloid progenitor 32D cell line was exposed to G-CSF for 7 days, mGC-1 expression was induced. Taken together, these results suggest that mGC-1 play an important role in granulocytic differentiation, and quite likely in mucousal immunity. To gain further insight into the potential pathways involved in GC-1 effects we performed further analysis in the 293 cell line. In situ hybridization showed mGC-1 is strongly expressed in the crypts of small intestine. hGC-1 cDNA has been transfected into 293 cells transiently and stably. In transfected 293 cells, hGC-1 was detected in the culture medium after 48h of transfection. In the Western blot, hGC-1 showed a multimer form in non-reducing gel, which was reduced to monomer after adding 10mM DTT. hGC-1 was also detected in the perinuclear region and cell surface.These data suggest hGC-1 is a secreted glycoprotein. This is consistent with tissue expression result that hGC-1 is only abuuundantly expressed in tissues which has secretion capability. Based on the data that hGC-1 is a glycoprotein and associated with cell adhesion, experiments have been performed to screen some lectins and cell adhesion proteins which might interact with hGC-1. Agarose bound lectin pull down assay shows hGC-1 strongly bound RCAI, weakly with ConA and WGA and no binding was detected with DSA, PNA and SNA. The purified hGC-1 enhances NIH3T3 and 293T/17 cell spread and attachment. hGC-1 enriched cell culture supernatants of 293T/17 cells transfected with hGC-1 expression vector also enhance cell spread and cell attachment. Co-immunoprecipitation demonstrated a association of hGC-1 with cadherin in 293 cells transiently transfected with hGC-1 cDNA. The interaction of hGC-1 with cadherin depends on the C-terminal olfactomedin domain, but does not require the five well conserved cysteine residues in hGC-1. However, cysteine residue at 83, 85, 246 and 437 is essential for hGC-1 secretion and cysteine 226 is critical for hGC-1 multimer formation. The potential association of hGC-1 with cadherin might explain the involvement of hGC-1 with cell adhesion. E-cadherin in particular, serves as a widely acting suppressor of invasion and growth of epithelial cancers, and its functional elimination represents a key step in the acquisition of the invasive phenotype for many tumors. Further analysis of the interaction of hGC-1 and the cadherin family of adhesion proteins may help clarify the role of this putative tumor suppessor gene.